Determination of erythromycins in fermentation broth using liquid phase extraction with back extraction combined with high performance liquid chromatography

AbstractLiquid phase extraction with back extraction (LPE-BE) combined with high performance liquid chromatography-diode array detection (HPLC-DAD) was applied for the extraction and determination of erythromycin A, B and C in fermentation broths. According to this procedure, the fermentation broth with the adjustment pH at a fixed value of 10 was first mixed with organic solvent (Vbroth/Vorg=1.0). After shaking, the mixture was separated into two phases by microfuging at 13,000rpm for 15min. Then back extraction was performed into the acidic aqueous phase with pH 5.0 (Vorg/Vaq=1.0). After centrifugation at 3000, the two phases were separated and 50μL of the acidic aqueous phase was injected into the HPLC. The effects of different variables such as the nature of extraction solvent and the pH of samples and buffer were investigated. At the most appropriate conditions, dynamic linear ranges of 0.5–8, 0.1–0.9 and 0.1–0.9mgmL−1 and limits of detection of 0.03, 0.003 and 0.002mgmL−1 were obtained for erythromycin A, B and C, respectively. Relative standard deviations (RSDs) of the proposed method were less than 9.5%. The mean recoveries were 99.5%. The proposed method is simple and sensitive with highly clean-up effect and it can be used for monitoring the progress of erythromycin fermentation